The broad objectives of this research are to determine how centrosomes and kinetochores function and interact to form the mitotic spindle in vertebrates, how the forces for chromosome motion are generated and regulated, and how progress through mitosis is controlled in animal cells. Aim #1 is to use 3-D EM tomography and immunogold methods to determine how spicen 300 is distributed relative to the gamma-tubulin-containing rings in isolated but functional centrosomes; Aim #2 is to determine if polar ejection forces are present in insect spermatocyte spindles, and to test the hypothesis that the final position a chromosome achieves on the spindle, just before anaphase onset, is determined by the number of non- kinetochore spindle Mts generated by the opposing spindle poles; Aim #3 is to evaluate the hypothesis that one kinetochore pulls while the other pushes during chromosome motion to the spindle equator (i.e., congression), and to determine the structural events that underlie congression of chromosomes containing only one kinetochore. Aim #4 is to evaluate the hypothesis at the direction a congressing chromosome moves is determined by the number of Mts on its sister kinetochores, and to complete our 3-D EM analyses on the structure of vertebrate kinetochores attached to Mts; Aim #5 is to test the hypothesis that the target for the inhibitor of anaphase onset produced by unattached kinetochores is associated with the spindle, and to determine if vertebrate cells also possess other checkpoint that regulate exit from mitosis; Aim #6 tests the hypothesis that the Mt minus-end motors associated with the kinetochore corona, which function during chromosome attachment, also function during anaphase; and Aim 7 is to test the hypothesis that the force-producing mechanism for centrosome separation during anaphase is active throughout mitosis. The knowledge obtained from these studies is needed to better understand the etiology of various birth defect syndromes and cancers, to design new therapeutic strategies for the control of cell proliferation, and for the treatment of other disease states including metastasis, arthritis and Alzheimer's.